Drosophila a Virus Is an Unusual RNA Virus with a T53 Icosahedral Core and Permuted RNA- Dependent RNA Polymerase

Journal of General Virology (2009), 90, 2191–2200 DOI 10.1099/vir.0.012104-0

Drosophila A virus is an unusual RNA virus with a T53 icosahedral core and permuted RNA- dependent RNA polymerase

Rebecca L. Ambrose,1 Gabriel C. Lander,2 Walid S. Maaty,3 Brian Bothner,3 John E. Johnson2 and Karyn N. Johnson1

Correspondence 1School of Biological Sciences, The University of Queensland, Brisbane, Australia Karyn N. Johnson 2Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA, USA [email protected] 3Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

The vinegar fly, Drosophila melanogaster, is a popular model for the study of invertebrate antiviral immune responses. Several picorna-like viruses are commonly found in both wild and laboratory populations of D. melanogaster. The best-studied and most pathogenic of these is the dicistrovirus Drosophila C virus. Among the uncharacterized small RNA viruses of D. melanogaster, Drosophila A virus (DAV) is the least pathogenic. Historically, DAV has been labelled as a picorna-like virus based on its particle size and the content of its RNA genome. Here, we describe the characterization of both the genome and the virion structure of DAV. Unexpectedly, the DAV genome was shown to encode a circular permutation in the palm-domain motifs of the RNA-dependent RNA polymerase. This arrangement has only been described previously for a subset of viruses from the double-stranded RNA virus family Birnaviridae and the T54 single-stranded RNA virus family Tetraviridae. The 8 A˚ (0.8 nm) DAV virion structure computed from cryo-electron microscopy and image reconstruction indicates that the virus structural protein forms two discrete domains within the capsid. The inner domain is formed from a clear T53 lattice with similarity to the b-sandwich domain of tomato bushy stunt virus, whilst the outer domain is not ordered icosahedrally, but forms a cage-like structure that surrounds the core Received 29 March 2009 domain. Taken together, this indicates that DAV is highly divergent from previously described Accepted 27 May 2009 viruses.

INTRODUCTION of the range and specificity of Drosophila responses to virus infection, it would be valuable to use viruses that naturally As obligate intracellular pathogens, viruses have complex infect Drosophila with a range of pathogenicities. interactions with their hosts. Comparatively little is known about the specific responses to virus infection in insects. During the 1970s, 11 viruses were described from Recently, insect responses that are specific for virus Drosophila (Brun & Plus, 1980; Gateff et al., 1980; Jousset infection have been investigated by using the genetically & Plus, 1975; Plus, 1978). Three of these, which were tractable Drosophila melanogaster (vinegar fly) model and isolated from D. melanogaster, were described as picorna- the most pathogenic of its viruses, the dicistrovirus like viruses based on morphological and biophysical Drosophila C virus (DCV) (Cherry & Perrimon, 2004; characteristics. The three viruses, DCV, Drosophila A virus Cherry et al., 2005; Deddouche et al., 2008; Ding & (DAV) and Drosophila P virus, were shown to be different Voinnet, 2007; Dostert et al., 2005; Galiana-Arnoux et al., viruses based on serology and sizes of the capsid proteins 2006; Hedges & Johnson, 2008; Hedges et al., 2008; (Jousset et al., 1972; Plus et al., 1976). These viruses are Roxstrom-Lindquist et al., 2004; Sabatier et al., 2003; natural pathogens of D. melanogaster, with about 40 % of Teixeira et al., 2008; van Rij et al., 2006) and the birnavirus wild populations being infected with one or more of these Drosophila X virus (Zambon et al., 2005). In future studies viruses (Plus et al., 1975). The three viruses have been shown to vary in their pathogenicity, with DCV being the The GenBank/EMBL/DDBJ accession number for the DAV genome most pathogenic and DAV the least. It is important to have sequence reported in this paper is FJ150422. an understanding of molecular characteristics of viruses to Two supplementary figures, showing alignments of the RdRP and the facilitate these studies (Huszar & Imler, 2008). Whilst DCV deduced capsid protein sequences of DAV with those of other RNA has been well-characterized and belongs to the genus viruses, are available with the online version of this paper. Cripavirus of the family Dicistroviridae (Christian et al.,

012104 G 2009 SGM Printed in Great Britain 2191 R. L. Ambrose and others

2005; Johnson & Christian, 1998), no detailed character- and stored at 220 uC until use. DAVHD virions were also isolated ization of the DAV genome or virion structure has been from infected DL2 cells by using a similar method after cells were reported. lysed by repetitive freeze–thawing at 220 uC. There are many small RNA viruses of insects that have Transmission electron microscopy (TEM) of purified virus. non-enveloped, 25–40 nm particles that encapsidate pos- Purified DAVHD was analysed by TEM as described previously itive-sense RNA genomes (Christian & Scotti, 1998; (Johnson & Ball, 2003). Fauquet et al., 2005). Of these, the majority with sequenced Nuclease treatment. For nuclease treatment, 1.2 mg DAVHD genomes are included in four taxonomic groups: the double-stranded (ds) RNA, prepared in vitro as described previously families Nodaviridae, Dicistroviridae and Tetraviridae and (Hedges & Johnson, 2008), and Flock House virus (FHV) RNA the floating genus Iflavirus (Fauquet et al., 2005). However, (Johnson et al., 2001) was incubated at 37 uC with 10 U RNase ONE in recent years, a number of viruses from insects have been (Promega) for 45 min. Samples were then diluted in RNA loading characterized that do not fit clearly into one of these buffer (Ambion), heated to 65 uC and separated on a non-denaturing 1 % agarose gel; products were visualized by staining with ethidium groups (Habayeb et al., 2006; Hartley et al., 2005; van der bromide. Wilk et al., 1997). Furthermore, within the family Tetraviridae, there are viruses with diverse genome cDNA synthesis, cloning and sequence determination. RNA was organizations that are classified together because of the extracted from sucrose gradient-purified DAVHD virions by using T54 symmetry of their capsid particles (Agrawal & TriReagent (Sigma-Aldrich) as per the manufacturer’s instructions, with Johnson, 1992; Gorbalenya et al., 2002; Gordon et al., the addition of 20 mg glycogen to increase RNA recovery. First- and 1995, 1999; Hanzlik et al., 1995; Pringle et al., 2001, 2003). second-strand cDNA was synthesized by using a SuperScript double- stranded cDNA synthesis kit (Invitrogen) with random hexamers as As more invertebrate RNA viruses are characterized fully, it recommended by the manufacturer. The cDNA products were ligated is becoming clear that there is a previously unrecognized into the EcoRV site of phosphatase-treated pBluescript SK+ (Stratagene) diversity of genome organization and expression strategies and sequenced. From these sequences, DAV-specific primers were within this group of viruses. These findings give us insight designed and used for further cDNA synthesis using SuperScript reverse both into virus evolution and into other biological transcriptase III (Invitrogen) as per the manufacturer’s instructions. processes (Koonin et al., 2008). 59 RACE (random amplification of cDNA ends) was performed as indicated by the manufacturer (Invitrogen) to obtain sequence at the 59 Here, we describe the molecular and structural character- end of the virus genome. A poly(A) tail was added to the 39 end of ization of DAV. The 30 nm particles encapsidate a purified DAVHD RNA by using poly(A) polymerase (Ambion), and the 39 positive-sense RNA that encodes two large open reading RACE system (Invitrogen) was used as per the manufacturer’s instructions to clone the 39 end of the DAV RNA. Nucleotide sequences frames (ORFs). The first of these has significant sequence were assembled and analysed by using DNASTAR software. similarities to the RNA-dependent RNA polymerases (RdRPs) of the tetraviruses and birnaviruses that have Cells. Drosophila DL2 cells (Schneider, 1972) were maintained at permuted RdRPs, whilst the second ORF encodes the 26 uC in Schneider’s medium (Invitrogen) supplemented with 10 % capsid protein. Analysis of the structure of DAV virions heat-inactivated fetal bovine serum and antibiotics. DL2 cells were 6 shows a unique capsid arrangement. seeded at 7610 cells per well into six-well tissue-culture plates and left to adhere at 26 uC overnight. Cells were infected with purified DAVHD virions in 500 ml additive-free Schneider’s medium or mock- infected and supplemented with 1.5 ml Schneider’s complete medium METHODS following incubation at 26 uC for 2 h. Infected cells were harvested at 0, 1, 2, 3 and 5 days post-infection (p.i.), washed in PBS and lysed in Propagation of DAV in D. melanogaster. A stock of Champetie`res either 1 ml TriReagent or 50 ml SDS-PAGE loading buffer. flies was obtained from the Drosophila Genetic Resource Centre of the Kyoto Institute of Technology, Japan (stock no. 103403), and a virus- SDS-PAGE and Western blot analysis. Virus and cell lysate free population was established essentially as described by Brun & Plus samples were heated at 99 uCin16 sample buffer for 5 min before (1980). The Australian isolate DAV was kindly provided by Dr Peter HD electrophoresis through SDS–10 % polyacrylamide gels at 170 V. Christian (Christian, 1992). DAV was diluted in sterile insect saline HD Alternatively, samples were loaded with sample buffer and a reducing (0.6 % NaCl, 0.04 % KCl, 0.024 % CaCl2 and 0.02 % NaHCO3)and agent on a NuPAGE 4–12 % Bis–Tris gradient gel in MES running injected into the haemocoel of virus-free D. melanogaster. Injected flies buffer (Invitrogen) and electrophoresed at 200 V. Proteins were were maintained on standard Drosophila medium (Sigma) for 10 days visualized by staining with Coomassie brilliant blue. at 25 uC and then frozen at 220 uC until purification. For Western blot analysis, separated proteins were transferred from Virus purification. DAVHD-infected flies were homogenized in SDS-PAGE gels to a nitrocellulose membrane and, following 50 mM Tris buffer, pH 7.4, and homogenates were centrifuged at blocking, were incubated with a 1/5000 dilution of an anti-DAV 5000 r.p.m. for 5 min to pellet fly debris. The virus was purified from antiserum overnight. Polyclonal antisera raised against both the the supernatant by pelleting through a 3 ml 10 % sucrose cushion at original French DAV isolate (Jousset et al., 1972) and the Australian 27 000 r.p.m. at 12 uC for 3 h in a SW41 swing-bucket rotor isolate DAVHD (Christian, 1992) were kindly provided by Dr Peter (Beckman). The resuspended virus was layered onto a continuous Christian. The membrane was washed then incubated with a 1/5000 10–40 % (w/v) sucrose gradient and centrifuged at 27 000 r.p.m. at dilution of a monoclonal anti-rabbit IgG conjugated with alkaline 12 uC for 3 h. The virus-containing fractions were harvested, then phosphatase for 2 h. Following three washes of the membrane in diluted in 50 mM Tris (pH 7.4) and virus was pelleted by TBST (Tris-buffered saline, Tween 20), bound antibody was centrifugation at 27 000 r.p.m., at 12 uC for 3 h. The virus was visualized with 0.5 % nitro blue tetrazolium and 0.25 % 5-bromo-4- resuspended in 50 mM Tris (pH 7.4) at 4 uC overnight, aliquotted chloro-indolyl phosphate (BCIP) in 1 M Tris buffer (pH 9.0). SeeBlue

2192 Journal of General Virology 90 Characterization of Drosophila A virus

II+ prestained markers (Invitrogen) were used to allow alignment of which provided a value of 8.32 A˚ (0.832 nm) resolution. Calculation proteins between Western blot and SDS-PAGE analysis. of the resolution by measure (Sousa & Grigorieff, 2007) at a 0.5 cut- off yielded a resolution of 8.69 A˚ (0.869 nm). Mass spectrometry. Peptide-mass fingerprinting and sequence analysis were conducted by using matrix-assisted laser desorption– ionization time-of-flight (MALDI-TOF) and electrospray ion trap RESULTS AND DISCUSSION (ESI-MS/MS) mass analysis, respectively. Samples were digested with modified trypsin (Promega) directly from solution or as gel pieces after SDS-PAGE. For MALDI-TOF, sinapinic acid was dissolved in DAV is a non-enveloped, icosahedral virus 50 % acetonitrile, 0.1 % trifluoroacetic acid (TFA) at half-saturation To visualize the morphology of DAV particles, sucrose concentration (saturated sinapinic acid solution was diluted with an gradient-purified virions were negatively stained and equal volume of the solvent) as matrix. A 1 ml aliquot of the DAVHD sample was mixed with 5 ml matrix; 1 ml of the mixed solution was examined by using TEM. At 6100 000 magnification, deposited onto a clean plate as one spot and the sample was analysed icosahedral, non-enveloped particles were observed, with a in a 4700 MALDI TOF/TOF system (Applied Biosystems) and a mean diameter of approximately 30 nm (Fig. 1a). Small Bruker Biflex III mass spectrometer. Linear mode was used above projections were visible on the virion surface in a regular 4000 m/z and reflectron mode below this threshold. Liquid array. The overall physical structure observed is consistent chromatography–tandem mass spectrometry (LC-MS/MS) used an with that of other insect RNA viruses (Johnson & Reddy, Agilent XCT-Ultra 6330 ion-trap mass spectrometer connected to an Agilent 1100 CapLC and ChipCube. Peptides were eluted and loaded 1998; Tate et al., 1999). Higher-resolution analysis is onto the analytical capillary column (43 mm675 mm inner diameter, discussed in greater detail below. also packed with 5 mm Zorbax 300SB-C18 particles) connected in line to the mass spectrometer with a flow of 600 nl min21. Peptides were eluted from a Zorbax 300SB-C18 system (Agilent) with a 5–90 % Structural protein analysis acetonitrile gradient (0.1 % formic acid) over 10 min. Data- To visualize DAV protein components, sucrose gradient- dependent acquisition of collision-induced dissociation MS/MS was purified virions were analysed by using SDS-PAGE (Fig. utilized. Protein identifications from peptide MS and MS/MS data were based on MASCOT (Matrix Science) searches of both an in-house 1b). A single major protein band was observed and the database containing all DAV ORFs and the GenBank non-redundant mass of this protein was estimated to be 42 kDa by database. Intact protein analysis was performed on a Bruker comparison with protein molecular mass standards. This micrOTOF ESI-TOF after a similar C18 reverse-phase separation. suggested that DAVHD has a single major capsid protein of Bruker MaxEnt deconvolution software was used to determine the approximately 42 kDa, which is similar to the estimated mass of the capsid protein. size of the major structural protein described previously for DAV (Plus et al., 1976). There was no evidence of the two Cryo-electron microscopy (cryoEM) analysis of particles. DAV was prepared for cryoEM analysis by preservation in vitreous ice over holey carbon films from Protochips (Cflats). Grids were cleaned prior to freezing by using a plasma cleaner (Gatan) using a mixture of 75 % (a) (b) (c) argon and 25 % oxygen for 10 s. A 3 ml aliquot of sample was applied kDa M DAV DAV to the grids, which were loaded into an FEI Vitrobot with settings at 70 4 uC and 100 % humidity. Grids were blotted for 4 s and stored at 60 liquid nitrogen temperatures. An Oxford 3500CT side-entry cryo- 50 stage was used for data collection. 40 * * Data were acquired by using a Tecnai F20 Twin transmission electron microscope operating at 120 keV, using a dose of approximately 19 30 2 ˚ 22 2 22 e A (0.19 e nm ) and a nominal underfocus ranging from 0.5 25 to 3.0 mm. In total, 2128 images were collected automatically at a 20 nominal magnification of 680 000 at a pixel size of 0.101 nm at the specimen level. All images were recorded with a Tietz F415 4K64K 15 pixel CCD camera (15 mm pixel) utilizing the Leginon data-collection software (Suloway et al., 2005). Experimental data were processed 10 with the Appion software package (Lander et al., 2009), which interfaces with the Leginon database infrastructure. The contrast transfer function (CTF) for each micrograph was estimated by using Fig. 1. Analysis of DAV virions and structural proteins. (a) Sucrose the Automated CTF Estimation (ACE) package (Mallick et al., 2005). gradient-purified virions were negatively stained and visualized by In total, 29 940 particles were selected automatically from the TEM at ¾100 000 magnification. Bar, 100 nm. (b) Proteins present micrographs by using a template-based particle picker (Roseman, in purified virus particles were separated by electrophoresis 2003) and extracted at a box size of 512 pixels. Only those particles through a 4–12 % Bis–Tris gradient gel. Total protein was whose CTF estimation had an ACE confidence of 80 % or better were visualized by staining with Coomassie blue. The capsid protein is extracted. Phase correction of the single particles was carried out indicated by an asterisk. Sizes of molecular mass markers are during creation of the particle stack. Stacked particles were binned by shown on the left. (c) Virion proteins separated through an SDS– a factor of two for the final reconstruction. The final stack contained 22 103 particles, with 17 063 contributing to the final density. The 10 % polyacrylamide gel were transferred to a nitrocellulose three-dimensional reconstruction was carried out by using the EMAN membrane and detected with a rabbit polyclonal anti-DAV reconstruction package (Ludtke et al., 1999). Resolution was assessed antibody. The band marked with an asterisk corresponds to the by calculating the Fourier shell correlation (FSC) at a cut-off of 0.5, 42 kDa protein indicated in (b). http://vir.sgmjournals.org 2193 R. L. Ambrose and others minor proteins of 32 and 73 kDa that were observed when (a)Cell lysate (b) Purified virus the original French DAV isolate was analysed by Plus et al. Mock DAV (1976). Western blot analysis was performed to analyse the DAV 0 5 0 1 2 3 5 M 12 kDa protein components in DAVHD, using an aliquot of the 60 original anti-DAV antiserum that was raised against the 50 French DAV isolate (Jousset et al., 1972). Three distinct * 40 * bands were detected by this analysis (Fig. 1c). The intense band indicated by the asterisk (Fig. 1c) migrates at a similar position to the 42 kDa protein observed in SDS-PAGE, showing that the 42 kDa protein is recognized by the Fig. 2. Detection of virus protein synthesis in infected Drosophila original DAV antiserum. Two additional bands were DL2 cells. (a) DL2 cells were either mock-infected or infected with detected at lower intensity and are observed consistently DAV and incubated at 26 6C until harvesting. Cell samples were in Western blot analyses of DAVHD virions. The molecular taken at 0, 1, 2, 3 and 5 days p.i. and lysed with sample buffer. masses of the minor proteins were estimated to be 50 and Cellular proteins were separated by electrophoresis through an 36 kDa for the larger and smaller bands, respectively. SDS–10 % polyacrylamide gel, transferred to a nitrocellulose Whilst these proteins were not observed in the SDS-PAGE membrane and incubated with an anti-DAV rabbit polyclonal gel shown in Fig. 1(b), they were seen by SDS-PAGE antiserum to detect virus proteins. The asterisk indicates the analysis when virus was overloaded (data not shown). It is 42 kDa capsid protein. Virus proteins were detected at 1 day p.i. possible that these additional proteins are analogous to the and their levels increased over time. No DAV-specific bands were two minor proteins described by Plus et al. (1976), detected in the mock-infected samples. (b) Proteins from DAVHD although the difference in size of the larger protein is virions purified from infected DL2 cells (lane 2) and adult difficult to reconcile. Unfortunately, no virus was recov- Drosophila (lane 1) were electrophoresed through an SDS– ered following passage of two different samples of the 10 % polyacrylamide gel stained with Coomassie blue. The original French DAV isolate (which had been stored at asterisk indicates the 42 kDa capsid protein. 220 uC for several decades) either in flies or in cell culture, so it was not possible to make direct comparisons between the isolates. The relationship between the capsid proteins was not clear from these data. However, the disappearance of the 50 kDa protein and correlated increase in the 42 kDa protein is DAV replicates in cell culture reminiscent of the cleavage of a capsid protein precursor. Furthermore, a small protein, estimated to be 6 kDa in To investigate whether DAV replication and assembly mass, was observed on SDS-PAGE gels overloaded for the occur in cell culture, Drosophila DL2 cells were infected major structural protein (Fig. 3). Many viruses encode with DAVHD and accumulation of capsid protein in cells their capsid proteins in a precursor polyprotein. Within the was analysed by Western blotting (Fig. 2a). Proteins that insect single-stranded (ss) RNA viruses, both nodaviruses co-migrated with each of the three proteins identified in and tetraviruses produce a capsid precursor protein that is purified virions were also detected in infected cells. No cleaved maturationally toward the C terminus to produce proteins were detected either in the mock-infected cells or the two mature capsid proteins (Johnson & Reddy, 1998). in the sample taken at time zero, the latter confirming that One interpretation of the DAV capsid protein profile is the input virus was below the level of detection in this that the 50 kDa protein is a capsid precursor protein, assay. All three DAV proteins were detected 1 day p.i. (Fig. which is cleaved during maturation of the virion to 2a) and the quantity of the proteins increased over time, produce the major 42 kDa capsid protein. If this is the case indicating that virus protein synthesis was occurring in cell for DAV, then the higher abundance of the 50 kDa protein culture. However, the ratio of the 50 and 42 kDa proteins in virus from DL2 cells may be due to incomplete cleavage was reversed compared with that observed in the purified of the precursor protein during maturation. However, in virion sample (Fig. 1b). To compare the ratio of proteins in contrast to nodaviruses and tetraviruses, no asparagine virus derived from cell culture with those in virus derived residue was identified in the DAV sequence as the potential from flies, virions were purified from both DL2 cells and cleavage site. Further analysis will be required to determine Drosophila by using the same method and the resulting the relationship between these proteins. The 36 kDa samples were analysed by SDS-PAGE (Fig. 2b). The virus protein may initiate from the second AUG of the capsid sample purified from DL2 cells contained two major ORF, as is observed in viruses of the family Nodaviridae proteins of similar abundance and the sizes corresponded (Johnson & Ball, 2003). to the 50 and 42 kDa proteins typically seen in Western blot analysis (Fig. 2b, lane 2). In contrast, virus purified Genome analysis from flies, as noted previously, contained only a single major 42 kDa protein (Fig. 2b, lane 1). The sucrose To analyse the nucleic acid encapsidated in virions, RNA gradient-purified virus from cultured DL2 cells was shown extracted from sucrose gradient-purified DAV was resolved to be infectious in naı¨ve DL2 cells (data not shown). on a 1 % agarose gel and nucleic acids were visualized by

2194 Journal of General Virology 90 Characterization of Drosophila A virus

M DAV extracted concurrently with the DAV sample was shown to kDa 220 be intact (Fig. 4). An alternative RNA-extraction kit 120 (Invitrogen) also did not improve the integrity of the 90 70 RNA (data not shown). Degradation of RNA purified from 60 50 tetravirus particles is common (Gordon & Hanzlik, 1998; 40 * Gordon et al., 1999; Pringle et al., 2003) and it is possible that removal of the genomic RNA from the virion results in 30 RNA degradation. We cannot discount other possible 25 explanations for the apparent diversity in size of the 20 encapsidated RNA. However, despite the smearing, an approximation of the maximum RNA size was inferred 15 from the distinct upper size cut-off of 5000 nt. 10 Nucleic acid was extracted from sucrose gradient-purified DAV and treated with RNase, resulting in degradation of the DAV genome (Fig. 4). Under the same conditions, the Fig. 3. Identification of a small structural protein. Virion proteins genome of the ssRNA virus FHV was also degraded, were overloaded and separated by electrophoresis through an whereas a synthetic dsRNA transcribed in vitro remained SDS–10 % polyacrylamide gel. Total protein was stained with intact (Fig. 4). This suggests that the genome of DAV is Coomassie blue. The asterisk indicates the 42 kDa capsid protein most likely to be ssRNA. band that is consistently observed in SDS-PAGE analyses. The arrow indicates the small protein band that is estimated to be By using a combination of approaches, clones correspond- approximately 6 kDa. ing to genomic RNA were synthesized and 4806 nt of genome sequence was determined. The sequences of all of the recovered clones formed a contiguous sequence, staining with ethidium bromide (Fig. 4). This showed a suggesting that DAV has a single genomic RNA. The 59 smear of RNA, which spanned sizes of approximately and 39 ends of the genomic RNA were targeted by using 1000–5000 nt compared with ssRNA markers; this size RACE. Two independent clones were identified with the range was confirmed by analysis on a denaturing same 59 end; however, it is possible that the genome formaldehyde agarose gel (data not shown). This suggested includes additional nucleotides at the 59 terminus. that the RNA was degraded during the extraction and Interestingly, 39 RACE was only successful when a synthetic electrophoresis processes, although the distinct edges of the poly(A) tail was added to the DAVHD RNA, indicating that smear and intact markers indicate that there was no RNase the DAV genome is not polyadenylated. contamination in the electrophoresis process. The RNA The genomic sequence was analysed for ORFs. The plus- smear was observed repeatedly, even when FHV RNA sense strand encoded two major ORFs and there were an additional three small ORFs encoding potential proteins containing .50 aa (Fig. 5). The 59-proximal ORF was the largest, encoding a putative protein of 1075 aa with a predicted molecular mass of 121 kDa. The 39-proximal ORF encoded 442 codons, which are predicted to comprise dsRNA DAV RNA FHV RNA M _ + _ + _ + a protein of 48.4 kDa. The second ORF initiates from a 10 000 methionine that is 43 nt downstream of the first ORF stop 8000 codon and is in the +1 reading frame compared with the 6000 4000 first. The 39 ORF overlaps the 59 coding region upstream of 3000 the initiating methionine (Fig. 5a). It is therefore possible 1500 that a +1 frameshift would allow synthesis of a 500 polyprotein including both large ORFs. However, +1 frameshifts are uncommon and it seems unlikely that the capsid proteins would be translated in this inefficient manner.

Fig. 4. Analysis of DAV genomic nucleic acid. RNA was extracted To identify proteins with sequence similarity to the deduced sequence of the 59-proximal ORF, the translated from purified DAVHD virions and mock-treated (”) or treated with RNase ONE (Promega) (+), which degrades ssRNA only. As sequence was used to search the non-redundant sequence controls, a synthetic dsRNA product from DCV and the ssRNA databases by using BLAST. Significant matches were found genome from FHV were treated similarly. Following nuclease to a large region of the RdRP proteins from two members treatment, products were separated on a 1 % agarose gel and of the family Tetraviridae and several members of the visualized by ethidium bromide staining. Sizes of the ssRNA family Birnaviridae. The highest similarity was found to the markers (M; in nt) are indicated on the left. RdRPs of Thosea asigna virus (TaV) and Euprosterna http://vir.sgmjournals.org 2195 R. L. Ambrose and others

To identify the structural protein-coding region, the sequence of the virion proteins was analysed. N-terminal sequencing of the structural protein was unsuccessful both from gel-purified protein and from whole virus, suggesting that the protein is blocked (data not shown). Protease mass mapping of virions in solution and of the 42 kDa capsid protein band after SDS-PAGE was tried next. By using a combination of MALDI-TOF and LC-MS/MS, seven and six peptides, respectively, spanning 29 % of the 39 ORF were identified, indicating that this ORF encodes the capsid protein (Fig. 5c). The predicted mass of 48.4 kDa is also consistent with the precursor protein band visualized on the protein gels (50 kDa). LC-MS/MS of purified particles produced a signal for the mature capsid protein of 40 480±3 Da. This does not match with a single cleavage event leading to capsid protein maturation as in insect nodaviruses and tetraviruses, both of which occur auto- catalytically at an asparagine residue (Johnson & Reddy, 1998). The fact that the mature protein mass does not match the amino acid sequence is, however, consistent with the N terminus being modified as discussed above; this will be investigated in future experiments.

Sequence-based fold recognition of the capsid Fig. 5. Organization of the DAV genome. (a) The 4806 nt DAV protein genome encodes two major ORFs and three additional small A BLAST search of the capsid protein’s amino acid sequence ORFs that contain .50 aa. The ORFs as shown start with an AUG against known insect virus genomes revealed a single codon; the extension (dotted line) on the capsid ORF indicates that this ORF overlaps the RdRP ORF, but the first methionine is homologue in the family Nodaviridae: 27 % identity was downstream of the RdRP ORF. (b) Analysis of the 59-proximal exhibited to the Wuhan nodavirus between residues 330 ORF showed high similarity of DAV RdRP regions to those of and 442. For further sequence-based analyses of the capsid members of the families Tetraviridae and Birnaviridae. These structure, various fold-recognition methods were utilized viruses have a permuted RdRP, with the GDD region upstream of via the structure prediction meta server (Ginalski et al., other palm subdomains. DAV RdRP also showed this permuted 2003), revealing a folding motif consistent with several arrangement, as indicated by C–A–B. (c) MS analysis indicates members of the families Tombusviridae [carnation mottle that the 39-proximal large ORF encodes the capsid protein. The virus (CMV), tobacco necrosis virus, tomato bushy stunt deduced sequence of the capsid protein is shown and the virus (TBSV)] and Sobemoviridae (cocksfoot mottle virus, peptides identified by MALDI-TOF (in bold type) and LC-MS/MS rice yellow mottle virus, southern bean mosaic virus) (see (underlined) are indicated. Supplementary Fig. S2, available in JGV Online). The results point to a conserved canonical b-sandwich fold beginning around residue 74 and ending around residue 236. Two viruses from the family Tombusviridae, CMV and elaeasa virus (EeV), which are closely related viruses from TBSV, whose structures contain a C-terminal domain that the family Tetraviridae. TaV and EeV both have an unusual protrudes from the capsid surface, additionally exhibited a permutation in the palm domain of the RdRP (Gorbalenya fold similarity to the DAV sequence between residues 270 et al., 2002). The nearly ubiquitous arrangement that is and 315. Conceivably, the N-terminal region of DAV forms observed in RNA viruses is that the GDD box (motif C) of an evolutionarily conserved b-sandwich domain while the the palm subdomain follows the conserved A and B motifs C-terminal domain forms a surface extension, much like (A–B–C) (Poch et al., 1989). However, in both TAV and the structures of CMV and TBSV. Indeed, cryoEM analysis EeV RdRPs, the C motif precedes the others in a C–A–B of the Malabaricus grouper nervous necrosis virus arrangement (Fig. 5b). This permuted RdRP is also (MGNNV), a fish nodavirus that appears to display a observed in some dsRNA viruses from the family similar domain organization upon sequence-based fold Birnaviridae, and structural analysis of the permuted recognition (Tang et al., 2002), revealed a C-terminal RdRP has shown a similar arrangement to the canonical domain that was separated from the b-sandwich domain RdRP of other viruses (Garriga et al., 2007; Gorbalenya et that formed the core capsid of the virus particle. The N- al., 2002). Interestingly, the RdRP of DAV also contains terminal portion of the sequence is highly charged, this C–A–B permutation (see Supplementary Fig. S1, including many arginines, which indicates that it may available in JGV Online). interact with the RNA and have a role in particle assembly

2196 Journal of General Virology 90 Characterization of Drosophila A virus

(a) (b) (c) Fig. 6. CryoEM analysis of DAV virions. (a) Representative electron micrograph of sucrose gradient-purified DAV in the frozen–hydrated state. (b) Averaged two-dimensional density Density corresponding to 22 000 centred raw particles. (c) One-dimensional radial plot of the 0 50100 150 200 averaged two-dimensional particles, showing Radius (Å) discrete peaks for the capsid layers.

(Tang et al., 2001; Venter et al., 2009). A closer look at the (Fig. 6a) exhibited a structural morphology that was very structure of DAV particles via cryoEM methods was reminiscent of MGNNV TEM images, whose radial density necessary to elucidate the overall structural morphology. distribution shows two distinct protein shells (Tang et al., 2002). Averaging of the computationally extracted and centred DAV particles from the micrographs confirms that CryoEM analysis of DAV particles the virus indeed appears to have a divided capsid shell (Fig. The structure of DAV was examined in detail by cryoEM 6b). From the selected particles, a three-dimensional structure image-reconstruction techniques. Interestingly, unpro- was computed to 8 A˚ (0.8 nm) resolution as estimated by the cessed, high-contrast cryoEM images of the virus particles FSC method, using a value of 0.5 (Fig. 7a, b).

(a) (b) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

0.1 0.2 0.3 0.4 0.5 _ Resolution (Å 1) (c) (d) Density Fig. 7. Analysis of three-dimensional reconstruction of DAV particles. (a) Surface repres- entation of the DaV cryoEM reconstruction 0 50 100 150 200 contoured at 2s. The inner N-terminal domain Radius (Å) (green) is well-ordered with visible secondary- (e) structural features, whereas the outer C- terminal domain (blue) contains only very- low-resolution features. (b) Estimated resolution of the reconstruction is 8 A˚ (0.8 nm) according to FSC criteria (y-axis) at 0.5. (c) Radially averaged slice through the centre of reconstruction. Blue, C-terminal domain; green, N-terminal domain; orange and red, RNA. (d) Plot showing density by radius [colours as in (c)]. (e) Docking of the b- sandwich domain of the TBSV crystal structure into the N-terminal domain of the DaV cryoEM density. http://vir.sgmjournals.org 2197 R. L. Ambrose and others

At these resolutions, the distribution of the capsid protein viruses, such as rotavirus (Dormitzer et al., 2004), these into two discrete and separate domains is plainly evident. domain extensions are dynamic and can undergo rearran- Viewed down the fivefold axis, the density distribution of gements during the virus life cycle. The cryoEM work the reconstructed particle shows the core domain extend- shown here provides evidence that DAV may be one such ing from a radius of 115 to 138 A˚ (11.5–13.8 nM) with a virus, whose dynamic outer domains, with possible maximum at 127 A˚ (12.7 nm), whilst the outer domain has biological function, are anchored to a well-ordered, b- a radius of 150–175 A˚ (15.0–17.5 nm) with a maximum at sandwich icosahedral core. 163 A˚ (16.3 nm) (Fig. 7c, d). These dimensions are similar to those of the MGNNV cryoEM structure, with two Conclusions additional layers of density corresponding to RNA, the outermost of which appears to interact directly with the DAV was first described as a picorna-like virus. The data capsid shell. Examination of the inner domain of the DAV presented here show that DAV is related evolutionarily to the electron density reveals an unmistakable T53 quasi- viruses with permuted RdRP core motifs in the families equivalent lattice, with many of the secondary-structural Tetraviridae and Birnaviridae. However, the capsid protein elements visible. Comparison of the core domain with the shows strongest sequence similarity to those of viruses in the b-sandwich domain of TBSV shows a very similar family Nodaviridae and conserved protein-folding motifs structural organization, and was docked into place by with the members of the family Tombusviridae.Inaddition, using an automated real-space rigid body refinement (Fig. the structure of DAV particles shows an inner T53core 7e). The docking shows that the network of subunit– surrounded by a less-well-defined outer protein domain. subunit interactions that make up the capsid is highly Taken together, these features show that DAV is an unusual conserved, and that capsid assembly in DAV is likely to be RNA virus with evolutionary relationships across a diverse similar to that of TBSV and other tombusviruses. Although range of invertebrate, fish and plant viruses. tombusviruses contain two distinct domains, it is note- ˚ worthy that they are not separated by the 10 A (1.0 nm) ACKNOWLEDGEMENTS gap present in the DAV structure. TBSV has a short polypeptide hinge connecting the N- and C-terminal We thank Peter Christian for providing virus and antisera and for domains, and it is probable that a similar, although much discussions, Darren Obbard (University of Edinburgh, UK) and Peter longer, hinge is seen in DAV. Christian for sharing unpublished data and Lopson Kebapetswe and Ivy Mui Tan for fly injections. This research was supported by a An unanticipated finding is the lack of highly ordered University of Queensland development grant to K. N. J. Electron structure corresponding to the outer domain. Whilst the microscopic imaging and reconstruction were conducted at the inner domain shows a detailed T53 organization, the outer National Resource for Automated Molecular Microscopy, which is supported by the NIH through the National Center for Research domain exhibits no such lattice, but only ambiguous Resources P41 Program (RR17573). B. B. acknowledges support from densities that appear to be an artefact of applying the National Science Foundation, MCB 0646499 and NIH grant no. icosahedral symmetry to a disordered or dynamic protein P20 RR-16455-06 from the Centers of Biomedical Research Excellence domain. Despite a lack of quasi-symmetry, however, the (COBRE) Program of the National Center For Research Resources. outer domain appears to be somewhat ordered, forming a B. B. also receives support from the Center for Bio-Inspired cage-like icosahedral lattice that surrounds the core Nanomaterials (Office of Naval Research grant no. N00014-06-01- 1016). We also thank the Murdock Charitable Trust for support of domain. The densities attributed to this dynamic C- the Mass Spectrometry Facility at Montana State University. terminal domain appear to converge above the icosahedral and quasi-twofold axes. In this regard, DAV diverges from the structure of MGNNV, whose outer domain consists of REFERENCES large protrusions of density at the quasi-threefold axis. Agrawal, D. K. & Johnson, J. E. (1992). Sequence and analysis of the Attempts to focus on this outer domain by masking out the capsid protein of Nudaurelia capensis v virus, an insect virus with interior region during the refinement did not provide any T54 icosahedral symmetry. Virology 190, 806–814. further insight into its organization, as the resulting Brun, G. & Plus, N. (1980). The viruses of Drosophila.InThe Genetics densities were equally ambiguous, with little deviation and Biology of Drosophila, pp. 625–702. Edited by M. 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